The tear film, ubiquitously present over the surface of the eye, is composed of an overlying lipid layer, a substantial middle aqueous component and an underlying mucous foundation. The mucous layer provides constant protection to the surface of the eye, and stability to the tear film. A rapid release of mucus in response to surface irritants, trauma, or toxins (bacterial and environmental) is necessary to replenish the mucous layer and protect the ocular surface.
Goblet cells of the conjunctiva are the primary source of mucus (complex glycoprotein) that constitutes the inner, mucous layer of the tear film. Regulation of normal goblet cell maturation and turnover, as well as goblet cell mucous synthesis and mucous secretion (mucous production), is important for the health of the ocular surface. In diseases such as keratoconjunctivitis sicca (KCS), Sjogren's Syndrome, vitamin A deficiency, anesthetic cornea, Stevens-Johnson Syndrome, thermal burns, chemical burns, cicatricial ocular pemphigoid, inactive trachoma, drug induced pseudopemphigoid, atopic diseases, radiation keratoconjunctivitis sicca, and superior limbic keratitis there is an alteration in goblet cell maturation, a disruption of mucous production, and a change in the mucous layer (41,49). Effective treatments for these diseases have not yet been developed because so little is known about the conjunctival goblet cell itself or about its secretory functions.
Goblet cells, which are highly polarized exocrine cells identified by their extensive apical accumulation of large secretory granules, are interspersed among the stratified epithelium of the conjunctiva. In the rat, goblet cells occur singly as well as in clusters of variable number (25,26,30,38,48). In humans, goblet cells generally occur singly, and, in addition, some goblet cells tend to group into mucous crypts of varying design in all areas of the conjunctiva (20,30). The intra-epithelial mucous crypts, in particular, closely resemble the clusters of goblet cells found in rat (30). The number of goblet cells per unit area (density) varies from bulbar to tarsal conjunctiva and from nasal to temporal areas in all species studied (25,26,30,38,48).
Goblet cells synthesize and secrete high molecular weight glycoproteins called mucins or mucus (11,50). When secreted, these mucins hydrate and gel, producing a protective mucous blanket covering the ocular surface. The mucous layer contains other components such as water, electrolytes, immunoglobulins (especially IgA), and enzymes. The non-mucin components of the mucous layer are secreted by the stratified squamous epithelial cells of the ocular surface and by the orbital glands. The mucous layer constitutes a physical and chemical barrier that protects the conjunctival and corneal epithelium from bacteria, from bacterial and environmental toxins, and from foreign bodies (37). The mucous layer stabilizes the tear film, prevents desiccation, and provides an optically smooth corneal surface by filling in surface irregularities (37), which is important for good visual acuity.
Little is known about the life cycle of the conjunctival goblet cell. It has been hypothesized, but not proven, that in the rat, goblet cells which form a cluster develop from a single stem cell of unknown location (26). These stems cells have not been found, nor has an immature conjunctival goblet cell been identified, as has been in the colon (70). Thus, the length of the goblet cell life span and the rate of goblet cell turnover have not been determined in the conjunctiva, although basal epithelial cells reach the conjunctival surface in 3-6 days (72). In the intestine, goblet cells mature from stem cells at the base of the crypt and live for 2-3 days (70). During this time, they migrate from crypt base to villus tip, a situation that does not occur in the conjunctiva. In the trachea, goblet cells may differentiate from serous epithelial cells (6) and their life span is longer.
Mature goblet cells display typical characteristics in all tissues studied, being large unicellular glands found in the surface of the epithelium and probably reaching to the basement membrane (72). They are connected by tight junctions to neighboring epithelial cells or other goblet cells and thus are polarized unicellular glands containing the biosynthetic enzymes for unidirectional synthesis and secretion of mucins. The synthetic pathway for mucin secretion by intestinal goblet cells has been demonstrated (50). The basal region of the cell contains the nucleus, mitochondria and rough endoplasmic reticulum (RER). The protein backbone of mucin is synthesized in the RER. The protein is then transported to the golgi apparatus, which is located above the nucleus. In the stacks of the golgi apparatus, the stepwise addition of carbohydrates to the protein backbone occurs as glycosyltransferases are compartmentalized in the golgi stacks (61). The synthesized mucins are then stored in condensed form in mucin granules that each are surrounded by membrane. The secretory granules fill the apical portion of the cell, and the large volume of apical secretory granules gives the goblet cell its distinct shape and appearance. Mucin secretion occurs by fusion of the mucin granule membrane with the apical plasma membrane, releasing the granule contents onto the ocular surface (67). Granule-granule fusion (compound exocytosis) can also occur and is the major type of fusion in stimulated secretion.
Two types of mucous secretion can occur, slow continual baseline or rapid accelerated secretion. In rabbit colon, in the absence of irritants or neurotransmitters, there is slow continual, baseline secretion (70) which represents periodic exocytosis of one or two mucin granules. Only a certain portion of the secretory granules participate in baseline secretion, those located on the periphery of the cell. It is not known what regulates this type of secretion. Nor is it known if this type of secretion occurs in conjunctival goblet cells or a population of conjunctival goblet cells.
In the intestine and colon, secretion of the entire goblet cell mucin contents can occur in a matter of minutes in response to a variety of stimuli (70). Secretion is an orderly series of membrane fusion events, which begins at the apical plasma membrane, proceeds first to the most central mucous granules and is then propagated to include peripheral mucous granules, and finally spreads to the most basal mucous granules until most of the granules have been secreted (67,70). This produces the cavitation typical of a stimulated intestinal epithelium. In this rapid secretion, the mucin granule membranes are not recycled, but are lost. The remaining goblet cell, however, maintains its shape and within 30 minutes the intracellular mucin is resynthesized (70,71). Again, little is known about the mechanism of mucous secretion in conjunctival goblet cell. A stimulated conjunctiva can have a cavitated appearance similar to that of the intestine, suggesting all or none secretion, but this has not been studied systematically. Nor is it known if conjunctival goblet cells resynthesize their mucins or instead are desquamated after secreting once.
Regulation of the mucous layer may require an extremely complex process, controlled at several different levels. A crucial question in the regulation of mucin secretion is what are the extracellular and intracellular signals that cause mucin granule fusion with the apical membrane. Baseline secretion and accelerated secretion appear to be regulated by different mechanisms. To date, the signals for baseline secretion are not known, but they have been identified for accelerated secretion in some tissues (70). In the intestine, electric field stimulation, parasympathetic nerves and muscarinic agonists stimulated crypt goblet cell mucin secretion (56,57,68). Because electric field stimulation of secretion was not completely blocked by the muscarinic antagonist atropine, a second, as yet unidentified, agonist exists that causes intestinal goblet cell secretion (57).
In addition to cholinergic agonists, neurotensin, which like cholinergic agonists causes an increase in intracellular [Ca.sup.2 +], increases intestinal goblet secretion (4). The role for agonists that increase intracellular levels of cAMP in stimulating intestinal goblet cell secretion remains controversial. Cholera toxin, which constitutively activates adenylate cyclase to produce cAMP, only indirectly stimulates mucin secretion, but the mediator is not known (60). The role of Vasoactive Intestinal Peptide (VIP), a cAMP-dependent agonist, varies between studies. In some intestinal or colonic goblet cell lines, VIP stimulates secretion (46); in some lines, VIP does not itself stimulate secretion, but potentiates the effect of cholinergic agonists (41); and finally in other cell lines and in vivo, it has no effect (42,51,60). Thus, in the intestine and colon cholinergic agonists and neurotensin, both of which increase intracellular [Ca.sup.2 +], appear to be the major stimuli of goblet cell mucin secretion. However, agonists such as VIP, which increase cAMP, appear to have a minor role, if any, in causing intestinal or colonic goblet cell secretion.
In the trachea, it has been shown recently that there are several different agonists that each stimulate goblet cell secretion. Cholinergic agonists using muscarinic receptors; ATP using purinergic, P.sub.2 receptors; Substance P, neurokinin A and neurokinin B using neurokinin NK.sub.1 receptors; calcitonin gene-related peptide; leukotriene D.sub.4 ; and platelet-activating factor each causes goblet cell secretion (14,22,31,35,59,78). The second messenger for these agonists is most likely Ca.sup.2+, although this has yet to be measured.
Studies performed to examine conjunctival goblet cell mucous secretion have been inconclusive. In one study mucin discharge was stimulated 2-3 fold by 8-Br-cGMP but was unaffected by 8-Br-cAMP (28). In another, 16,16-dimethylprostaglandin E.sub.2 (dmPGE.sub.2), which can increase cAMP levels, was shown by electron microscopy to cause fusion of individual conjunctival goblet cell mucin granules and subsequent discharge of their contents onto the ocular surface (3,13). However, it has also been shown that high [K.sup.+ ], which is known to cause neural stimulation in tissues in general, does not stimulate goblet cell mucous secretion. Physical manipulation of the eye itself will cause the conjunctival goblet cells to secrete mucus (13).
Normal maturation of goblet cells and normal goblet cell mucin production are important for the health of the ocular surface. Either an increase in mucous secretion and/or in goblet cell maturation, or a decrease in mucous secretion and/or in goblet cell maturation can cause ocular surface problems. An increase in mucus in the tear film occurs in vernal conjunctivitis, giant papillary conjunctivitis, and irritation or injury to the ocular surface (2). A decrease in mucus or change in the character of mucus in the tear film occurs in diseases such as keratoconjunctivitis sicca (KCS), SoSgren's Syndrome, vitamin A deficiency, anesthetic cornea, Stevens-Johnson Syndrome, cicatricial ocular pemphigoid, inactive trachoma, thermal burns, chemical burns, drug induced pseudopemphigoid, atopic diseases, radiation KCS, and superior limbic keratitis (16,17,32,41,49,51,66). In KCS and Sjogren's Syndrome, there is a decrease in goblet cell number and an increase in mucus strands in the tear film (41); however, the presence of such strands indicates decreased clearance of mucus and not increased mucous secretion.
That both an increase or a decrease in the mucous layer can disrupt the ocular surface suggests that the mucous layer is tightly regulated. This regulation could be occurring at many levels--from the central nervous system, to the conjunctival epithelium (including goblet cell and stratified squamous cell secretion), to the goblet cell itself, to the tear film. Currently, there are no ways to clinically evaluate the mucous layer, mucin secretion, mucin synthesis, or goblet cell maturation to determine at which level the regulation has broken down, creating the disease process. The availability of methods to evaluate the state of conjunctival goblet cells and of methods of either stimulating or inhibiting mucous secretion as appropriate would go far in providing ways of diagnosing and treating ocular injuries or diseases and maintaining the health of the ocular surface.